lecture_notes:mar31
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| * Ion Torrent | * Ion Torrent | ||
| - | == Pyrosequencing == | + | == Pyrosequencing (SBS) == |
| When a nucleotide is added to the template chain by a polymerase, a PPi is released which is converted to an ATP by one enzyme. In the presence of ATP another enzyme, luciferase, releases light. You record when the light was released (ie which nucleotide was added to the plate at that time) and also the intensity of the light. The light intensity tells you how many nucleotides were added in a row. | When a nucleotide is added to the template chain by a polymerase, a PPi is released which is converted to an ATP by one enzyme. In the presence of ATP another enzyme, luciferase, releases light. You record when the light was released (ie which nucleotide was added to the plate at that time) and also the intensity of the light. The light intensity tells you how many nucleotides were added in a row. | ||
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| The packing beads that are placed around the bead which holds the template strands in the individual wells on the plate are what hold the various enzymes (other than the polymerase) needed for the reaction. | The packing beads that are placed around the bead which holds the template strands in the individual wells on the plate are what hold the various enzymes (other than the polymerase) needed for the reaction. | ||
| - | == solid == | + | == solid (Hybridization) == |
| - | == Illumina/solexa == | + | 4 colors and 1024 possible 8mer fragments. |
| - | == Ion Torrent == | + | Each round gives the nucleotide identity at the first two positions of the 8mer fragment with a 3 nucleotide gap between each two nucleotides. Ie one run gives you ...---AB---CD---... You then perform multiple runs which start in the -1 position from the previous run so eventually when you combine all of these runs you get the entire fragment sequence. |
| + | |||
| + | Each of these images takes several hours, so a whole run may take several days. | ||
| + | |||
| + | |||
| + | == Illumina/solexa (SBS) == | ||
| + | |||
| + | Rather than running emulsion PCR on beads to amplify a fragment into a colony of identical fragments on a single bead, you wash fragments over a slide and hope they stick to the slide far apart from eachother. You can then amplify these fragments on the slide which forms the dense single sequence identity colony you need to get a good read. | ||
| + | |||
| + | Rather than sequencing single nucleotides at a time (and having the possibility for a series of single nucleotides to be sequenced at a time) you sequence all nucleotides (ACGT) simultaneously and have each nucleotide type give off a different color. You then get the identity of one nucleotide from the entire plate, and move on to the next nucleotide on the whole plate. | ||
| + | |||
| + | Theoretically you could sequence very long fragments this way, but practically the quality starts degrading too much after about 50 bases. | ||
| + | |||
| + | == Ion Torrent (SBS) == | ||
| + | |||
| + | Works very similarly to pyrosequencing, except rather than requiring light from luciferase enzymes it takes advantage of the proton that is released when a nucleotide is added to a template. It measures this electric current. The presense of a current over a certain template when a specific nucleotide is added to solution tells you that that nucleotide was added to that template. The level of current at a template tells you the number of nucleotides that were added in that run. | ||
| + | |||
| + | Can sequence about 60 bases every 100 minutes. | ||
| + | |||
| + | Nader recommended reading the 2006 PNAS paper for background on this technology. | ||
| === Ideas for data storage layout in Campusrocks === | === Ideas for data storage layout in Campusrocks === | ||
| + | |||
| + | Everything is stored in: | ||
| + | |||
| + | /campusdata/BME235/ | ||
| + | |||
| + | Kevin proposed having the following folders. | ||
| + | |||
| + | bin/ | ||
| + | bin/scripts/ | ||
| + | bin/x86_64 | ||
| + | bin/[picky_program_directories] (store programs that require the proximity of helper scripts) | ||
| + | data/ | ||
| + | programs/ (store program source and test programs before adding them to bin) | ||
| + | lib/ | ||
| + | experiments/ (store trial runs while testing out settings etc) | ||
| + | |||
| + | There should be a README in each directory describing the downstream directories, the purpose, and the contents. | ||
| + | |||
| + | |||
| + | Kevin stressed that Makefiles are a good way of generating reproducible, and fairly complex runs. Anyone who needs more background on makefiles should see the gnu make documentation which is freely available online. Also the various subfolders of ~karplus/pluck/ have examples of makefiles Kevin has used for his work with assembling genomes. | ||
| + | |||
| + | |||
| + | Also we meet in the science library this Friday. | ||
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lecture_notes/mar31.txt · Last modified: 2010/04/01 10:44 by svasili
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